Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure, but also directly contributes to lung injury and augments established acute lung injury (ALI), a syndrome known as ventilator-induced lung injury (VILI). Molecular mechanisms underlying VILI remain unclear and effective pharmacotherapy has yet to emerge. Our genomic-intensive approaches (murine, canine, human ALI models) captured pre-B cell colony enhancing factor (PBEF) as a novel ALI susceptibility candidate gene and novel biomarker in sepsis and ALI. Furthermore, we determined that PBEF promoter SNPs were significantly associated with susceptibility to sepsis and ALI and ventilator-free days and ALI mortality. Intratracheally-administered rhPBEF increased PMN recruitment into the murine alveolar space, increased lung expression of PMN chemoattractants, and was highly synergistic with mechanical ventilation in producing increases in BAL protein, PMN's, and inflammatory cytokines. Heterozygous PBEF mice (with targeted deletion of a single PBEF allele) were protected from severe murine VILI. Bioinformatic analyses revealed strong PBEF-driven induction of VILI gene ontologies (NF-(B, leukocyte extravasation, oxidative stress, ROS). Complicating this seemingly tight pathobiologic scenario is the observation that in addition to strong proinflammatory cytokine properties of extracellular PBEF, it is also normally present as an intracellular nicotinamide phosphoribosyltransferase (NamPT) with key enzymatic activity involved in nicotinamide adenine dinucleotide (NAD) biosynthesis. As PBEF appears to be an essential participant in the development of VILI, Specific Aim 1 will functionally characterize key regulatory elements which drive VILI-mediated PBEF expression (mechanical stress- and ROS-responsive promoter elements, 5' SNPs). Specific Aim 2 will explore exogenous PBEF involvement in NF-(B-dependent innate immunity pathways in lung endothelium, possibly via Interferon-Induced Transmembrane 3 protein, IFITM3, a novel PBEF binding partner that we identified from bacterial two-hybrid studies. Strongly supported by identification of other novel PBEF-binding partners (NAD Dehydrogenase Subunit 1, Ferritin Light Chain, Apoptotic Protease Activating Factor-1), Specific Aim 3 will examine the role of intracellular PBEF in regulation of apoptosis and ROS responses in lung EC. Finally, Specific Aim 4 will refine therapeutic strategies designed to limit PBEF's influence in VILI (neutralizing antibody, siRNA). We will assess the involvement of NamPT activity in homeostasis using enzyme inhibitors. In addition to providing novel therapeutic VILI strategies, these studies will yield novel mechanistic insights into VILI-associated pathobiology and elucidate PBEF influences on ALI/VILI susceptibility, severity, and mortality. PUBLIC HEALTH RELEVANCE: Acute lung injury (ALI) is a devastating inflammatory lung disease with an estimated 190,000 cases/yr in the United States and an unacceptable mortality rate of 30-40%. Mechanical ventilation directly contributes to de novo lung injury and exaggerates established ALI, a syndrome known as VILI [4-5]. Despite recent advances in care of the critically ill (2), there remains a need for improved understanding of ALI/VILI pathophysiology and a need to improve upon the severely limited therapeutic options currently available for these patients. Using a systems biology-like approach (genes to populations), we have identified PBEF as a novel biomarker and candidate gene in ALI with genetic variants associated with sepsis and ALI susceptibility